Re: CNBr cleavage
Daniel Hess (Daniel.Hess@fmi.ch)
Tue, 31 Mar 1998 08:49:45 +0200
>I was wondering if anyone can offer some advice on CNBr cleavages. I have
>been trying to cleave my protein by adding a small CNBr crystal to my
>protein in 70% TFA, but I'm not sure I am having much success. Either my
>protein is not cleaved, or I think I am getting too many fragments when I
>analyze my cleaved protein with MALDI-TOF. I usually just put my sample in a
>small tube and put this is a dark drawer for 16-18 hours.
>
>I would also greatly appreciate it if anyone can help me with the following
>questions.
>
>How long does a CNBr cleavage really take? I have read in the literature it
>is anywhere from 4-24 hours.
>
>Do I need to remove the oxygen from my tube (covering with argon for
>example), or is capping the tube sufficient?
>
>Can adding too much CNBr lead to cleavages at other sites?
>
>Is TFA the best choice to use considering I will be analyzing my protein on
>a MALDI?
>
>Can 70% TFA lead to acid based cleavages by itself, and can formic acid
>really lead to a reduction of disulfide bonds? ( I read a message about the
>formic acid on this discussion group a while ago)
>
>Thanks a lot in advance
>
>
>___________________________________________
>NC State University
>Department of Biochemistry
>Phone (919)515-5786
>Fax (919)515-2047
Dear Brett,
try cleavage in 0.1M HCL. You will get less unwanted side reactions like
desamidation, formulations etc. You also get better cleavage of Met-Ser and
Met-Thr (Kaiser et al. had a Poster at the ABRF, 98). One problem can also
be that your Met are oxidized and therefor not cleaved. A MS of the intact
protein could tell you that.To much CNBr could also lead to oxidation of
Met. Take a 50x molar excess of CNBr. Measure the weight of a CNBr crystall
in the hood, add 0.1M HCl, take an alliquot out, add to Protein, cover with
Ar, cleave in the dark over night.
If your protein is not soluble in 0.1M HCL you can add up to 6M UREA, clean
it on a mixed bed ionexchanger first!
with best regards Daniel
Dr. Daniel Hess
Friedrich Miescher Institut
Postfach 2543
CH-4002 Basel
Tel.: 061 697 85 44 or 061 697 68 68 FAX: 061 697 3976
e-mail: dhess@fmi.ch